Intestinal muscle undergoes stretch intermittently during peristalsis and persistently proximal to obstruction. The influence of this pervasive biomechanical force on developing smooth muscle cell function remains unknown. We adapted a novel in vitro system to study whether stretch modulates transforming growth factor-β1 (TGF-β1) and type I collagen protein and component α1 chain [α1(I) collagen] expression in fetal human intestinal smooth muscle cells. Primary confluent cells at 20-wk gestation, cultured on flexible silicone membranes, were subjected to two brief stretches or to 18 h tonic stretch. Nonstretched cultures served as controls. TGF-β1 protein was measured by ELISA and type I collagen protein was assayed by Western blot. TGF-β1 and α1(I) collagen mRNA abundance was determined by Northern blot analysis, quantitated by phosphorimaging, and normalized to 18S rRNA. Transcription was examined by nuclear run-on assay. Tonic stretch increased TGF-β1 protein 40%, type I collagen protein 100%, TGF-β1 mRNA content 2.16-fold, and α1(I) collagen mRNA 3.80-fold and enhanced transcription of TGF-β1 and α1(I) collagen by 3.1- and 4.25-fold, respectively. Brief stretch stimulated a 50% increase in TGF-β1 mRNA content but no change in α1(I) collagen. Neutralizing anti-TGF-β1 ablated stretch-mediated effects on α1(I) collagen. Therefore, stretch upregulates transcription for TGF-β1, which stimulates α1(I) collagen gene expression in smooth muscle from developing gut.